We have used the combination of single-molecule Fö rster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Fö rster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a ␤-structure content of the collapsed unfolded state of Ϸ20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.Gaussian chain ͉ microfluidic mixing ͉ protein folding ͉ random coil ͉ secondary structure W ith the discovery of small proteins that fold in the absence of populated intermediates (1), our quantitative understanding of the elementary properties of protein folding reactions has made significant advances, including the structural characterization of transition states for folding (2) and the prediction of folding rates from native structure (3-5). One of the most severe limitations for the further development of these approaches is our ignorance about the energetic or structural properties of unfolded † † states of proteins. Because of the structural heterogeneity and complexity of the ensembles of conformations populated by unfolded proteins, their experimental characterization has proven extremely difficult. Traditional methods, such as small-angle scattering techniques (6), provide only global physical properties, e.g., the radius of gyration. In some cases, more detailed structural information can be obtained from NMR (7-10), but these studies usually provide information about the denatured state only under nonnative conditions, typically in the presence of large concentrations of denaturant, or through severe destabilization of the native state induced by covalent modification or mutations. The most interesting and physiologically relevant situation, however, is that of an unfolded sta...

No abstract

Photoionization (PI) of Fe14+ in the range from 450 to 1100 eV was measured at the BESSY II storage ring using an electron beam ion trap achieving high target-ion area densities of 10(10)  cm(-2). Photoabsorption by this ion is observed in astrophysical spectra and plasmas, but until now cross sections and resonance energies could only be provided by calculations. We reach a resolving power E/ΔE of at least 6500, outstanding in the present energy range, which enables benchmarking and improving the most advanced theories for PI of ions in high charge states.

We have investigated the lowest binding-energy electronic structure of the model cuprate Sr 2 CuO 2 Cl 2 using angle-resolved photoemission spectroscopy. Our data from about 80 cleavages of Sr 2 CuO 2 Cl 2 single crystals give a comprehensive, self-consistent picture of the nature of the first electron-removal state in this model undoped CuO 2-plane cuprate. First, we show a strong dependence on the polarization of the excitation light which is understandable in the context of the matrix element governing the photoemission process, which gives a state with the symmetry of a Zhang-Rice singlet. Secondly, the strong, oscillatory dependence of the intensity of the Zhang-Rice singlet on the exciting photon energy is shown to be consistent with interference effects connected with the periodicity of the crystal structure in the crystallographic c direction. Thirdly, we measured the dispersion of the first electron-removal states along ⌫→(,) and ⌫→(,0), the latter being controversial in the literature, and have shown that the data are best fitted using an extended tJ model, and extract the relevant model parameters. An analysis of the spectral weight of the first ionization states for different excitation energies within the approach used by Leung et al. ͓Phys. Rev. B 56, 6320 ͑1997͔͒ results in a strongly photon-energy dependent ratio between the coherent and incoherent spectral weight. The possible reasons for this observation and its physical implications are discussed.

We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 micros. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible.